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Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1728-1734
Partial Tandem Duplications of the MLL Gene Are Detectable in
Peripheral Blood and Bone Marrow of Nearly All Healthy Donors
By
Susanne Schnittger,
Bernhard Wörmann,
Wolfgang Hiddemann, and
Frank Griesinger
From the Department of Hematology and Oncology, University of
Göttingen, Göttingen, Germany.
 |
ABSTRACT |
Partial tandem duplication within the MLL gene has recently been
described as a novel genetic alteration in acute myeloid leukemia
(AML). It has been associated with trisomy of chromosome 11, but was
also identified in AML patients with normal karyotypes. The current
study was performed to investigate whether MLL duplications are
restricted to AML, and hence whether they may also occur in normal
hematopoietic cells. MLL-duplication transcripts were analyzed by
nested reverse-transcriptase polymerase chain reaction (RT-PCR) in
peripheral blood in two groups of 45 and 20 patients, respectively, as
well as in two bone marrow samples from healthy volunteers. Duplications were detected in two independent nested RT-PCR experiments in the peripheral blood samples of 38 of 45 (84%) and 20 of 20 (100%)
of the two groups and in both bone marrow samples. On this basis, MLL
duplications seem to occur frequently in a subset of cells in normal
hematopoiesis. The type of partially duplicated MLL transcripts varied
substantially. Three transcripts were identical to those known from
AML. In addition, four new transcripts were characterized. Three of
these four were in frame and potentially translatable. MLL duplications
were also detected by seminested genomic PCR with intron 9- and intron
1-specific primers in 20 of 20 peripheral blood samples studied,
indicating that the duplications are genomically fixed at the DNA level
and are not an RT-PCR artifact. In summary, MLL duplications are
regularly generated by homologous ALU recombination in a small number
of hematopoietic cells of most or even all healthy donors. These data
suggest that MLL duplications are not implicated in the malignant
transformation in AML, or alternatively, that only a few cells will
acquire additional oncogenic mutations necessary to establish the
malignant phenotype of AML.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
CHROMOSOMAL rearrangements are thought to
play a crucial role in the development of hematologic malignancies.
These disorders arise from a single transformed cell by a multistep
process that involves sequentially occurring mutations of genes, which
may function as oncogenes or tumor-suppressor genes.1-3
Specific chromosomal alterations have been found in association with
certain morphologic subtypes of acute myeloid leukemia (AML), as
t(8;21) in a subset of M2, and less often in M1,4-6
t(15;17) in M3,7 and inv(16)8 in M4eo.
Rearrangements of the MLL gene by reciprocal translocations involving
chromosome band 11q23 are well described in infants and adults with AML
and acute lymphocytic leukemia (ALL), and involve more than 30 chromosomal regions as translocation partners.9,10 About
half of the partner genes have been cloned to date. Rearrangements are
strikingly diverse, involving partner genes, with various predicted
protein functions like transcription factors (AF4,11
AF9,12 AF10,13,14 AF17,15
ENL10,16), RNA polymerase II elongation factor
(ELL17), cell-cycle regulation factor (CBP18),
forkhead protein family members (AFX19), or SH3 domain
family member EEN,20 or seem to be target genes for RAS
such as AF6.21,22 In contrast to the diversity of the partner genes, all translocations occur within a well-defined breakpoint cluster region restricted to an 8.3-kb genomic area of the
large 100-kb MLL gene23 encompassing exons 8 to 14 (nomenclature by Nilson et al, 199624). In addition,
partial tandem duplications of the NH2 region of this part
of the gene have been associated with trisomy of chromosome 11 in
AML,25 and recently, have also been reported in AML
patients with normal karyotypes.26-29 The chimeric
transcripts code for proteins with potential oncogenic activity. The
mechanism by which the partial tandem duplication contributes to
leukemogenesis is currently unknown, as is the mechanism of all
rearrangements involving MLL. It is still unclear whether MLL-fusion
genes act as oncogenes or whether the fusion proteins have dominant
negative effects. Introduction of an MLL-AF9 transgene resulted in
expansion of the myeloid compartment, and after a median interval of 8 months, the development of AML.30 Retroviral transduction
of murine progenitor cells with an MLL-ENL transgene leads to
immortalization and establishment of myeloid leukemias after several
passages in mice hosts.31 These data suggest a gain of
function mechanism for MLL-fusion genes and demonstrate that additional
events are probably necessary for the establishment of the leukemogenic
phenotype. Other data are more consistent with the possibility that
loss of function of the MLL gene is important in
leukemogenesis.32 Thus, the MLL gene may be a
tumor-suppressor gene. However, as MLL-deficient embryonic stem cells
are inable to differentiate normally, it is highly suggestive that MLL
is involved in the regulation of early stages of hematopoietic
development, either directly or indirectly. The MLL translocations in
ALL are most commonly associated with a pre-pre-B
phenotype,33-35 whereas AMLs with MLL translocations are
predominantly seen in the monocytic subtypes.36 The MLL
duplication has been found in most AML subtypes37,38 and
does not seem to be restricted to specific cell lineages. If a mutation
does not fatally affect cells, it might be observed throughout the
normal life of an individual. In the current study, we addressed the
question whether MLL tandem duplications can be detected via a nested
reverse-transcriptase polymerase chain reaction (RT-PCR) in blood
samples of healthy donors. We were able to show that MLL duplications
occur frequently in a small number of cells in healthy individuals. In
addition, our data strongly suggest that MLL duplication transcripts
are not splicing artifacts, but most likely result from homologous Alu
recombination of the MLL gene, similar to that described for
AML.25,26
| |
MATERIALS AND METHODS |
Probands.
Normal individuals gave informed consent and the procedure was approved
by the Ethics Committee of the University of Göttingen as of June
14, 1996. Equal numbers of males and females were analyzed. The mean
age was 45.6 (range, 7 to 78). A description of the AML patients used
as controls in this study will be given elsewhere (Schnittger et al,
in preparation).
Nonhuman specimens.
For dilution experiments to determine the level of sensitivity of
detection of MLL-duplication transcripts, cDNA was prepared from
peripheral blood mononuclear cells of Maccaca mulatta, rat kidney, as well as mouse (strain SJL) liver and spleen. Tissue was
mechanically dispersed to single-cell suspension for RNA extraction and
processed as described later.
Nucleic acid isolation.
DNA was extracted with a salting-out procedure39 from
peripheral blood cells after Ficoll separation of mononucleated cells. Total RNA was isolated from peripheral blood or bone marrow after Ficoll separation with RNeasy (Quiagen, Hilden, Germany) following the
manufacturer's instructions. PolyA+ mRNA was separated from total RNA
using Oligotexll particels (Quiagen).
RT-PCR.
One microgram total RNA was reverse-transcribed with 200 U Superscript
(GIBCO-BRL, Eggenstein, Germany) in a 40-µL reaction using random
primers. cDNA equivalent to a quantity of 25 ng reverse-transcribed RNA
was amplified for 35 cycles (1 minute at 94°C, 1 minute at 63°C,
and 1 minute at 72°C), in 50 µL with 10 pmol of each primer (3.C1 and MLLint), 10 mmol dNTPs, and 1.25 U Taq
polymerase (GIBCO-BRL) in the buffer shipped by the supplier. Nested
PCR was performed with an aliquot of 1 µL of primary PCR reaction
with primers 6.1 and E3AS (Fig
1). Nucleotide sequences of the primers
were as follows: 3.1C, 5 AGGAGAGAGTTTACCTGCTC3
(bp 821 to 840); MLLint, 5CTTCCAGGAAGTCAAGCAAGCAGGT3
(bp 3869 to 3892); E3AS, 5ACACAGATGGATCTGAGAGG3 (bp 567 to
586); and 6.1, 5GTCCAGAGCAGAGCAAACAG3 (bp 4013 to 4032). The
positions of RT-PCR primers within the MLL gene are given in Fig 1,
with sequence numbering according to Tkachuk et al.10
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| Fig 1.
Exon structure of the 5 region of the MLL gene. Old and
new nomenclature (Nilson et al,24) of the exons is
indicated. Positions of the primers used for RT-PCR are given below the
schematic exons.
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For each RNA sample, an ABL-specific RT-PCR was performed to control
the integrity of RNA using primers abl5 GGCCAGTAGCATCTGACTTTG and abl3 ATGGTACCAGGAGTGTTTCTCC. cDNA of an AML with MLL
tandem duplication was used as a positive control and water instead of cDNA was included as a blank sample in each experiment.
Peripheral blood from 45 healthy donors was analyzed in a first
screening and an additional 20 samples (isolated by caesium chlorid
gradient centrifugation and kindly provided by Dr Reis from the
Institute of Human Genetics) in a second screening with completely new
reagents and performed in a different laboratory building. In this
laboratory, no PCR experiments with MLL as a target gene had ever been
performed before this series of experiments. In addition, two bone
marrow samples from patients with no hematologic malignancies were
analyzed.
Genomic PCR.
Seminested PCRs were performed with the same reaction conditions as for
RT-PCR with 100 ng of genomic DNA using primers MLLi6 (5GCTGAGATAGAAGGATTGTCTTG3 [bp 998 to 1020]40) or
MLLi8 (5GTCCCAATAATTCCTTTATGGC3 [bp
5963-5985]40), and MLLi1p
(5AGAGTCAGGTGGCTAACTG3)41 as external primer in the first
35 cycles. One microliter was further amplified with nested primer
MLLi1n 5CTCCTCTTCAAAGACATCTG3 (bp 571 to 590; Genbank U66259) for 35 cycles. Both amplifications were performed at 55°C.
Sensitivity.
One microgram of RNA was isolated from approximately 100,000 cells and
was reverse-transcribed in one experiment. As we used 1/40 µL of the
RT reaction for PCR, we analyzed an equivalent of 25,000 cells per
sample. The amount of DNA used for genomic seminested PCR corresponded
to approximately 10,000 mononucleated cells.
Dilution experiments with cDNA from a duplication-positive AML and a
normal donor sample were performed in 10 1 steps up to
108. To equalize the total amount of cDNA in the
reaction mixture, cDNA prepared from mouse spleen was added. Mouse
spleen RNA was found to be negative for MLL-duplication transcripts.
Sequencing.
RT-PCR products were separated on 2% agarose gels, cut from the gels,
and isolated with Quiaex II (Quiagen) following the manufacturer's
instructions. Approximately 100 ng of purified RT-PCR products was
directly sequenced with 3.3 pmol of primer 6.1 for forward and
E3AS for reverse reactions using the Dye Terminator Cycle
Sequencing Kit (Perkin Elmer, Weiterstadt, Germany). After initial denaturation at 95°C for 5 minutes, 25 cycles at 94°C for
15 seconds and 60°C for 4 minutes were performed. Sequence analysis
was performed on 6% polyacrylamid gels on an ABI 373 sequencer.
| |
RESULTS |
MLL duplication transcripts are detected by nested RT-PCR in peripheral
blood of 84% to 100% of normal donors.
In all 45 normal blood samples, no MLL duplication transcripts were
detectable after 35 cycles of the primary PCR (data not shown). After
an additional 35 cycles with nested primers, various amplified
transcripts were detected in 38 of 45 samples (84.4%) (Fig
2). In a second series of experiments in a
physically distant laboratory building with completely new reagents,
RNA MLL-duplication transcripts were amplified in 20 of 20 samples,
probably due to better quality RNA (Fig 3).
The sensitivity of the second experiment was 1 log more sensitive than
the first series. In the first series, MLL-duplication transcripts were
detected solely in undiluted cDNA, whereas in the second series,
dilutions of proband material into mouse cDNA at a concentration of
101 still showed the duplication transcripts (data not
shown).
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| Fig 2.
Ethidium bromide-stained agarose gel with
nested RT-PCR products of normal donor blood (samples 1 to 8 and 26 to
34).  K, blank control; +K, AML with MLL duplication; M, molecular
weight standard (Boehringer no. VI). Exon fusions are indicated at the
left and right. New characterized fusions are underlined.
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| Fig 3.
Nested RT-PCR products of the second experiment. (A) ABL
PCR; (B) nested PCR for MLL duplication AML with MLL duplication. M,
molecular weight standard (Boehringer no. VI); C, blank control;
+C, AML with MLL-duplication; C46-65, pheripheral blood of healthy
controls no. 46 to 65.
|
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Dilution experiments to determine the sensitivity of nested RT-PCR
for the detection of MLL-duplication transcripts.
For dilution experiments, tissues of several nonhuman species were
tested. Peripheral blood of two individual Maccaca mulatta showed MLL-duplication transcripts with the identical length of an MLL
exon 9/exon 3 fusion transcript as in an AML (+C, Fig
4). The 200-bp amplification product was
sequenced and showed an exon 9/exon 3 fusion. The nucleotide sequence
was exactly identical to the human 200-bp fragment. In contrast, no
MLL-duplication transcripts were detected in rat kidney, mouse spleen,
and liver specimens. Therefore, mouse liver cDNA was used for dilution
experiments. cDNA of an AML with an exon 9/exon 3 fusion transcript was
diluted into mouse cDNA at concentrations ranging from 100
to 108. The MLL duplication was detected up to a
concentration of 105 (Fig
5). Thus, the frequency of MLL-duplication
transcripts in peripheral blood of healthy individuals is estimated at
one in  5,000 circulating cells, as 25,000 cells were included per
reaction and each sample showed an average of five amplification
products.
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| Fig 4.
cDNA of AML positive for an exon 9/exon 3 duplication
transcript (+C), peripheral blood of Maccaca mulatta animals
no. 5 and 8 (M5, M8), rat kidney (RK), and mouse liver and spleen (ML,
MS) was amplified for MLL-duplication transcript. Description of other
lanes as in Fig 2.
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| Fig 5.
cDNA of AML positive for an exon 10/exon 3 duplication
transcript was diluted into mouse liver cDNA at concentrations ranging
from 100 to 105 (0 to 5) and
MLL-duplication transcripts were amplified by nested PCR. For each
experiment, cDNA of ~25,000
cells was used. Description of other lanes as in Fig 2.
|
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Sequence analysis of MLL-duplication transcripts.
Sequencing of RT-PCR products showed fusions of exons 9, 10, and 11, with exon 3 identical to those found in AML
patients.25-27,38 In addition, several new fusion products
were identified. Four of these fusions were characterized by sequencing
and showed MLL exon 9/exon 4 and exon 9/exon 3, as well as exon 13/exon
3 fusions. One fusion transcript included a cryptic pseudoexon from
intron 1 fused to exon 11 (Figs 2 and 6).
All transcripts, with the exception of the exon 9/exon 4 fusion, are in
frame and potentially translatable. In addition, several yet
uncharacterized transcripts were identified (Figs 2 and 3). In the
second experiment with high-quality RNA, more than one transcript was
detected in all samples (Fig 3). Most samples showed four to six
different transcripts. The exon 9/exon 3 fusion transcript, which has
been associated with AML, was found to be the most common form (24 of
65 cases). Others, like the large transcript with fusion of exon 11 to
a cryptic pseudoexon from intron 1 followed by exon 3 could only be
found in single cases. To rule out an RNA-processing artifact in
precursor RNA, we isolated mRNA from the total RNA of two normal bone
marrow samples and peripheral blood of eight additional normal donors. Amplification products were obtained indicating that at least exon
9/exon 3 and exon 10/exon 3 fusion transcripts, as well as transcripts
not observed in AML, were processed to mRNA and thus are potentially
functional.
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| Fig 6.
Schematic presentation of the exon fusions (new
nomenclature after Nilson et al24). AML specific fusions at
the left and new characterized fusion at the right. All fusions can be
found in peripheral blood of healthy donors.
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Genomic MLL duplication in peripheral blood of healthy individuals.
To evaluate whether MLL duplications occur at the genomic level,
genomic seminested PCRs for the MLL duplication using intronic primers
complementary to introns 1, 9, and 11 were performed. Specific
amplification products (Fig 7) identical to
those found in AML with MLL duplication were obtained. Sequencing of
these products showed Alu sequences (data not shown). This suggests that the duplications are present at the DNA level and are probably Alu-mediated recombinations. Thus, MLL duplications in healthy donors
do not seem to be a mechanism at the transcription level or an artifact
by RT-PCR. In addition, Southern blots of BamHI and
EcoRI with a 0.74-kb MLL cDNA probe encompassing exons 8 to 10 and 12 to 15 (Oncor, Heidelberg, Germany)23
only showed germline bands in normal controls, whereas AML samples
positive for the duplications all showed additional fragments
indicative of MLL rearrangements (data not shown). This is consistent
with a higher amount of cells with the duplications in AML versus
healthy donors. Our data strongly suggest that MLL duplications occur
in a subset of cells in normal hematopoiesis at the genomic level and
that MLL-duplication transcripts are detectable in total RNA, as well as in processed mRNA.
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| Fig 7.
Genomic amplification of the duplications using intron 9 and intron 1 primers. M, molecular weight standard; C, blank
control; +C, AML with MLL duplication.
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| |
DISCUSSION |
The present study provides convincing evidence that partial tandem
duplications within the MLL gene are not restricted to malignant cells
in AML, but may also occur in a subset of normal hematopoietic cells.
Using a highly sensitive nested RT-PCR approach, duplications within
the MLL gene were identified in 38 of 45 and 20 of 20 peripheral blood
and two bone marrow samples of healthy donors in two separate sets of
analysis. This alteration seems to be a relatively frequent event, as
it can be detected in an RNA equivalent of approximately one in 25,000 cells. We found various forms of transcripts, of which all but one are
potentially translatable. Genomic PCR data indicate that the MLL
duplication transcripts result from MLL duplications at the genomic
level and are not a transcriptional or RT-PCR artifact. Furthermore, contamination was formally ruled out by a second series of experiments performed in a physically distant institute with completely new reagents and RNA.
The presence of tumor-associated fusion genes in normal donor blood has
been described for the BCL2-IgH transcript specific for germinal center
lymphoma with t(14;18) and the BCR-ABL fusion gene, the hallmark of
t(9;22)-positive chronic myeloid leukemia. BCL2-IgH transcripts have
been shown to be present at a low level, but are detectable with nested
RT-PCR in 50% of normal donors.42-44 These findings point
to a low-level transcription rate or alternatively to a small
subpopulation of cells carrying the translocation t(14;18). Similarly,
at least one t(9;22)-specific BCR-ABL fusion transcript in
108 cells has been found in a 30% of healthy
donors.45,46 For both translocations, the incidence of
fusion transcripts increases with age. It has been postulated that
these translocations are first hits in tumor development and that
additional genetic alterations are necessary to establish the malignant
phenotype. The incidence of MLL duplications in healthy donors seems to
be much higher than that of BCL2-IgH or BCR-ABL, as MLL duplications
are detectable in nearly all samples and with a less sensitive PCR
technique as that described for BCL2-IgH and BCR-ABL. In addition, more than one partially duplicated transcript is usually detected, making up
to an average of about 5 different duplications per 25,000 cells.
Thus, the estimated mean number of duplication-positive cells in
healthy blood donors is 1 in 5,000 cells, strongly suggesting that
the duplication is continuously generated during normal hematopoiesis. Furthermore, new, not previously described transcripts were
characterized by sequencing, and genomic fusions were identified by
PCR. It should be stressed that in addition to new duplication
transcripts, the most frequent duplication transcript is the MLL exon
9/exon 3 fusion, which is also found in AML. The genomic data on the basis of PCR showed identical fragments in healthy donors as in AML.
Sequencing of the genomic PCR fragments showed Alu sequences at or near
the breakpoints. However, the exact fusion sites were not identified,
because genomic fusions resulted into reconstitution of intact ALU
sequences. These data are consistent with the hypothesis of homologous
Alu recombination within the MLL gene occurring at a relatively high
level in most or even all normal individuals. The biologic significance
of MLL duplications in healthy individuals is currently unclear. It may
be speculated that these transcripts have a special function in a
specific subtype of cells. Alternatively, the MLL duplications may
represent preleukemic alterations such as the BCL2-IgH and BCR-ABL
fusions, and further alterations are required for the induction of the
leukemic phenotype. Whether the predominance of only three duplication
transcripts in AML is a result of selection for the duplication
transcript with higher transforming potential is currently unknown.
Another hypothesis may be that highly recombinogenic structures within
the MLL gene, probably the frequently occurring ALU repeats, may lead
to frequent alterations within the gene. Homologous ALU recombination,
leading to partial gene duplication or deletion, has been shown to be implicated in the genesis of a number of nonmalignant
diseases.47 A unique feature of partial gene duplication is
that the duplicate copy is usually organized in tandem with the
original copy in a head to tail orientation.46 Unequal
crossing over has generally been accepted as the underlying mechanism.
Some of these rearrangements were found to occur within Alu
elements,48,49 as was described for duplications within the
MLL gene in AML.25,26 In any case, the MLL gene seems to be
a target for homologous recombination of Alu repeats in normal
individuals and may serve as a model to study Alu recombination in
genetic disorders.
Furthermore, our data demonstrate that nested PCR for MLL duplication
is not suitable for the detection of minimal residual disease, as has
been recently suggested by Satake et al.50 MLL duplication
is obviously not a new molecular target for monitoring minimal residual
disease in patients with AML and normal karyotype.
| |
NOTE ADDED IN PROOF |
After submission of this article, two additional reports by Marcucci et
al51 and Caldas et al52 described MLL
duplication transcripts in healthy individuals.
| |
FOOTNOTES |
Submitted December 8, 1997;
accepted April 24, 1998.
Supported by a program grant of the Deutsche Forschungsgemeinschaft to
F.G. and B.W. (SFB500, project A1).
Address reprint requests to Susanne Schnittger, PhD,
Department of Hematology/Oncology, University of Göttingen,
Robert Koch Str. 40, 37075 Göttingen, Germany.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We greatly acknowledge Dr J. Reis (Institute of Human Genetics,
Göttingen) for providing RNA samples. Professor Dönecke (Institute of Biochemistry, Göttingen) is acknowledged for
providing laboratory space and equipment for the second series of
experiments.
| |
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Abstract
Introduction
Abstract